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1. Atlanta E. coli data, 2019-2023

   https://doi.org/10.4211/hs.30600d19187f4a0cb3489908e07e652d  

   Ledford, Sarah (August 2025, HydroShare)

   Urban streams and rivers have chronic bacteria contamination in the United States, coming from multiple sources, following a variety of flowpaths to the waterway, and with differing downstream fates. Bacteria from human sewage, estimated through measures of Escherichia coli, pose the highest risk to human health. We analyzed four years of E. coli monitoring by community science groups to look for spatial and temporal drivers of E. coli densities in watersheds in the urban core of metro Atlanta, GA, with a wide range of racial and economic diversity as well as persistent patterns of segregation and racialized inequality. These watersheds are spaces of environmental injustice, with disproportionate impacts for lower-wealth and predominantly Black communities from flooding, soil contamination, and air pollution. While there were minimal differences in E. coli between watersheds with different Black and white populations, individual sites could be identified as hot and cold spots of contamination. Storm events increased E. coli at most sites, indicating a combination of runoff and sediment-sorbed E. coli explains about 50% of the temporal variability in E. coli densities. Long-term median E. coli levels were not strongly correlated to land cover or socio-demographic characteristics of the contributing watershed, but E. coli variability was lower in less densely urbanized areas. Temporal and spatial distributions of E. coli are controlled by complex interactions between sources and hydrologic transport that vary across watersheds. While direct correlations to racial demographics were not observed, the interactions between sewage as one environmental harm and the many others (air quality, soil quality, prison-industrial complex, etc.) present in minority and low-income urban communities emphasize the oversized burden environmental justice communities carry. 

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2. Crystal structure of E. coli PRPP synthetase

   https://doi.org/10.1186/s12900-019-0100-4  

   Zhou, Weijie; Tsai, Andrew; Dattmore, Devon A.; Stives, Devin P.; Chitrakar, Iva; D’alessandro, Alexis M.; Patil, Shiv; Hicks, Katherine A.; French, Jarrod B. (December 2019, BMC Structural Biology)
3. Structure of the E. coli agmatinase, SPEB

   https://doi.org/10.1371/journal.pone.0248991  

   Chitrakar, Iva; Ahmed, Syed Fardin; Torelli, Andrew T.; French, Jarrod B. (April 2021, PLOS ONE)

   Permyakov, Eugene A. (Ed.)

   Agmatine amidinohydrolase, or agmatinase, catalyzes the conversion of agmatine to putrescine and urea. This enzyme is found broadly across kingdoms of life and plays a critical role in polyamine biosynthesis and the regulation of agmatine concentrations. Here we describe the high-resolution X-ray crystal structure of the E . coli agmatinase, SPEB. The data showed a relatively high degree of pseudomerohedral twinning, was ultimately indexed in the P 3 1 space group and led to a final model with eighteen chains, corresponding to three full hexamers in the asymmetric unit. There was a solvent content of 38.5% and refined R/R free values of 0.166/0.216. The protein has the conserved fold characteristic of the agmatine ureohydrolase family and displayed a high degree of structural similarity among individual protomers. Two distinct peaks of electron density were observed in the active site of most of the eighteen chains of SPEB. As the activity of this protein is known to be dependent upon manganese and the fold is similar to other dinuclear metallohydrolases, these peaks were modeled as manganese ions. The orientation of the conserved active site residues, in particular those amino acids that participate in binding the metal ions and a pair of acidic residues (D153 and E274 in SPEB) that play a role in catalysis, are similar to other agmatinase and arginase enzymes and is consistent with a hydrolytic mechanism that proceeds via a metal-activated hydroxide ion. 

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4. Molecular organization of the E. coli cellulose synthase macrocomplex

   https://doi.org/10.1038/s41594-021-00569-7  

   Acheson, Justin F.; Ho, Ruoya; Goularte, Nicolette F.; Cegelski, Lynette; Zimmer, Jochen (March 2021, Nature Structural & Molecular Biology)

   null (Ed.)
5. Increased cytoplasmic expression of PETase enzymes in E. coli

   https://doi.org/10.1186/s12934-024-02585-w  

   Carter, Luke_M; MacFarlane, Chris_E; Karlock, Samuel_P; Sen, Tridwip; Kaar, Joel_L; Berberich, Jason_A; Boock, Jason_T (November 2024, Microbial Cell Factories)

   Abstract BackgroundDepolymerizing polyethylene terephthalate (PET) plastics using enzymes, such as PETase, offers a sustainable chemical recycling route. To enhance degradation, many groups have sought to engineer PETase for faster catalysis on PET and elevated stability. Considerably less effort has been focused toward expressing large quantities of the enzyme, which is necessary for large-scale application and widespread use. In this work, we evaluated severalE. colistrains for their potential to produce soluble, folded, and activeIsPETase, and moved the production to a benchtop bioreactor. As PETase is known to require disulfide bonds to be functional, we screened several disulfide-bond promoting strains ofE. colito produceIsPETase, FAST-PETase and Hot-PETase. ResultsWe found expression in SHuffle T7 Express results in higher active expression ofIsPETase compared to standardE. coliproduction strains such as BL21(DE3), reaching a purified titer of 20 mg enzyme per L of culture from shake flasks using 2xLB medium. We characterized purifiedIsPETase on 4-nitrophenyl acetate and PET microplastics, showing the enzyme produced in the disulfide-bond promoting host has high activity. Using a complex medium with glycerol and a controlled bioreactor,IsPETase titer reached 104 mg per L for a 46-h culture. FAST-PETase was found to be produced at similar levels in BL21(DE3) or SHuffle T7 Express, with purified production reaching 65 mg per L culture when made in BL21(DE3). Hot-PETase titers were greatest in BL21(DE3) reaching 77 mg per L culture. ConclusionsWe provide protein expression methods to produce three important PETase variants. Importantly, forIsPETase, changing expression host, medium optimization and movement to a bioreactor resulted in a 50-fold improvement in production amount with a per cell dry weight productivity of 0.45 mg_PETaseg_CDW⁻¹ h⁻¹, which is tenfold greater than that forK. pastoris. We show that the benefit of using SHuffle T7 Express for expression only extends toIsPETase, with FAST-PETase and Hot-PETase better produced and purified from BL21(DE3), which is unexpected given the number of cysteines present. This work represents a systematic evaluation of protein expression and purification conditions for PETase variants to permit further study of these important enzymes. 

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